The present invention relates to the use of HPPD inhibitors as selection agents in the transformation of plant cells and of plants by genetic engineering. The transformation of plant cells and of plants by genetic engineering generally consists in introducing a foreign or heterologous gene encoding a protein of interest into the genome of the plant cells or of the plants which contain them. Once it has integrated into the genome of the plant cells, this heterologous gene is then expressed so as to confer on said cells and on said plants which contain them a novel characteristic related to the function of the heterologous gene which is expressed.
Many techniques for transforming plant cells and plants by genetic engineering have been developed and thoroughly described in the literature. Firstly, methods which seek to introduce a DNA fragment carrying the heterologous gene in the form of xe2x80x9cnaked DNAxe2x80x9d may be distinguished. This involves, in particular, bombarding cells, protoplasts or tissues with particles to which the DNA sequences are attached. Other methods may be used, such as microinjection or electroporation, or alternatively direct precipitation using PEG. Secondly, methods consisting in using, as a means of transfer into the plant, a heterologous chimeric gene in an agrobacterium tumefaciens Ti plasmid or an agrobacterium rhizogenes Ri plasmid will be distinguished. Those skilled in the art will choose the suitable method depending on the nature of the plant cell or of the plant to be transformed. Mention will in particular be made of the following Patents and Patent Applications: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159, EP 604 662, EP 672 752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174, EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 and WO 95/06128.
The methods for transforming plant cells generally comprise the following steps:
a) preparing competent plant cells capable of receiving the heterologous gene in a suitable medium,
b) transforming the competent cells with the heterologous gene,
c) growing and selecting the transformed cells comprising the heterologous gene in a suitable medium.
The competent plant cells may be embryogenic calluses, cell cultures on a solid support or in suspension, or embryogenic tissues, which are well known to those skilled in the art and widely described in the literature.
The production of transgenic plants, comprising the heterologous gene integrated into their genome, then consists in carrying out the following steps of:
d) regenerating plants from the transformed cells in one or more suitable media and, where appropriate,
e) producing and recovering the seeds of the fertile transformed plants.
The pollination of the regenerated plants in order to produce the seeds of the fertile transformed plants takes place either by self-pollination or by cross-pollination with a nontransformed variety of the same plant or, optionally, with another variety which has stably integrated another heterologous gene into its genome.
The seeds of the transformed plants are then used in conventional selection programmes in order to produce novel varieties of transgenic plants which have stably integrated the heterologous gene into their genome. Such selection programmes are well known to those skilled in the art and comprise evaluating the agronomic properties of the plants produced and of their descendants, in particular with respect to the agronomic properties related to the expression of the heterologous gene.
The transformed cells are selected using a selection marker gene. Such marker genes and their use in transforming host organisms are well known to those skilled in the art and are widely described in the literature.
Among the genes encoding selection markers, mention may be made, firstly, of the genes encoding easily identifiable enzymes such as the GUS enzyme (or GFP, xe2x80x9cGreen Fluorescent Proteinxe2x80x9d), and genes encoding pigments or enzymes which regulate the production of pigments in the transformed cells. Mention will be made, secondly, of the genes for resistance to antibiotics and the genes for tolerance to herbicides (bialaphos, glyphosate or isoxazoles). In this case, the selection takes place by introducing into the medium suitable for the growth and selection of the transformed cells a selection agent of the antibiotic or herbicide type which is lethal for the nontransformed cells, only the cells comprising the gene for resistance to antibiotics or to herbicides being capable of growing on the selection medium. Such selection marker genes are in particular described in Patent Applications EP 242 236, EP 242 246, GB 2 197 653, WO 91/02071, WO 95/06128, WO 96/38567, WO 97/04103 or WO 99/24585.
The selection marker genes are introduced into the host cells simultaneously with the heterologous gene, either in the same vector, the two genes being associated in a convergent, divergent or colinear manner (WO 95/06128, U.S. Pat. No. 5,731,179), or in two vectors used simultaneously for transforming the plant cells. Under certain conditions (U.S. Pat. No. 5,731,179), and in particular when the heterologous gene and the selection marker gene are introduced separately in two vectors, simultaneously, the heterologous gene encoding a protein of interest and the selection marker gene may integrate on two different chromosomes in the genome of the transformed plant. It is possible, after recovering fertile transformed plants, to eliminate the marker gene in order to produce transformed plants comprising only the heterologous gene encoding a protein of interest. This elimination takes place by self-fertilization or by crossing the transformed plants comprising the heterologous gene and the selection marker gene with a nontransformed variety of the same plant, the segregation of the two genes occurring in conventional Mendelian fashion.
When the heterologous gene encoding a protein of interest is a herbicidal tolerance gene, the heterologous gene alone may be used as the selection marker in the process for transforming the plant cells or the plants.
The use of genes for tolerance to herbicides which are HPPD inhibitors, as selection markers in the processes for transforming plant cells and plants, has been described in the literature (WO 96/38567, WO 99/24585). The HPPD inhibitor is introduced into the culture medium of the cells after transformation (step c), in the same way as the other selection agents, according to the usual practices of those skilled in the art. HPPD inhibitors act on plant cells by inhibiting the synthesis of plastoquinones and of carotenoids. This action produces a bleaching of the plant cells which is not harmful to the growth of said cells, more particularly in the case of embryogenic tissues. Only the transformed plant cells comprising the gene for tolerance to HPPD inhibitors remain green and can be selected since they thus differ from the nontransformed cells.
The present invention consists in improving such a use in such a way as to facilitate the process for identifying and selecting the transformed cells. A second object of the present invention consists in decreasing the time required for selecting the transformed plants and for producing fertile regenerated plants. Specifically, the general process for transforming, selecting, regenerating and recovering the seeds of fertile transformed plants may take several months depending on the plants under consideration, about 10 to 18 months in particular for plants such as soya bean. Decreasing this duration by one or more months constitutes a definite technological and economical advantage.
The present invention consists in introducing the HPPD inhibitor into the culture medium of the competent plant cells (step a) so as to bleach said cells before the transformation step. The bleached competent cells are then transformed with the gene for tolerance to HPPD inhibitors, as a selection marker, and the transformed cells which have integrated said selection marker into their genome become green, enabling them to be selected. Such a process makes it possible to decrease the time required for selecting the transformed cells by several months, about 2 to 3 months.
The present invention therefore consists of a method for transforming plant cells by introducing a heterologous gene into said plant cells with a gene for tolerance to HPPD inhibitors as selection markers, said method comprising the steps of:
a) preparing and culturing competent plant cells capable of receiving the heterologous gene in a suitable medium,
b) transforming the competent cells with the heterologous gene and the selection marker,
c) growing and selecting the transformed cells comprising the heterologous gene in a suitable medium,
characterized in that a step for bleaching the competent plant cells is carried out before the transformation step (b), by introducing a suitable amount of HPPD inhibitor into the suitable culture medium of the competent plant cells.
The present invention also consists in producing transgenic plants comprising the heterologous gene integrated into their genome, and then consists in carrying out the following steps of:
d) regenerating plants from the transformed cells selected in one or more suitable media and, where appropriate,
e) producing and recovering the seeds of the fertile transformed plants.
Preferably, the transgenic plants produced using the method according to the invention are fertile transgenic plants.